ABI3 REGULATION Streszczenie raportu

Caspases are a family of cysteine-dependent aspartate-specific proteases that play a prominent role in apoptotic cell death in mammalian systems. Upon a pro-apoptotic stimulus, 'intitator caspase' proteins get activated by cleavage into a p10 and a p20 subunit. These activated initiator caspases cleave the downstream 'effector caspases', that in turn cut a wide range of apoptotic substrates, resulting in the Programmed cell death (PCD) of the cell. In plants, PCD shares the morphological features with mammals, the molecular mechanisms however are largely unknown. In the Arabidopsis thaliana genome, nine caspase like genes are present, named Metacaspases (MCs). Upon the presence of a N-terminal 'prodomain', metacaspases 1 to 3 were classified as Type I and metacaspases 4 to 9, which lack this domain, as Type II. In analogy to the mammalian caspases, recombinant Type II MCs show cysteine-dependent autocatalytic processing. Although, the two tested Type II MCs were arginine/lysine-specific proteases in contrast to their mammalian homologues.

In this project, it was decided to focus on the Type I metacaspases. The N-terminal extension reminds of the pro-domain present in mammalian initiator caspases. Type I MCs contain an LSD1 protein motif in their 'prodomain', previously shown to be present in two proteins related in cell death in plants. Metacaspase 1 (MC1) shows induction after infection with pathogens that cause the hypersensitive response, a type of PCD that prevents spreading and proliferation of the pathogen. The focus of this project is the study of MC1 in A. thaliana.

1) Polyclonal antibodies (Abs) against MC1 and LSD1 were raised in rabbit. 2) A T-DNA insertion line available at the Max Planck Institute (Gabi-KAT) had an insertion in the first intron of the MC1 gene, and was confirmed as a complete knock-out. A SALK knock-out line for LSD1 in A. thaliana Col-0 background was identified and showed uncontrolled spreading of lesions under normal growth conditions. 3) To test the effect of constitutive or conditional higher expression of MC1 protein levels, transgenic plants were produced. Full length MC1, full length MC1 with the putative active cysteine mutated into a serine, the N-terminal part containing the prodomain and the C-terminal caspase domain were overexpressed under the 35S constitutive or B-estradiol inducible promoters. Transformed lines with higher MC1 expression levels were identified using the affinity purified polyclonal antibodies. Several lines overexpressing full length MC1 and MC1 C-terminal caspase domain were identified. Though, no lines overexpressing the N-terminal domain only could be found. Instability due to the truncation of the protein could be the cause, since mRNA levels in certain lines were clearly increased. 4) The different available transgenic lines for Metacaspase 1 (MC1 knock-out line and overexpression lines) were analysed under normal growth conditions and with the PCD inducing agent Fumonisin B1 (FB1). No differences in phenotytype were observed. Apparently there is a redundancy issue playing here. 5) A transgenic A. thaliana line with MC1-GUS under control of MC1 native promoter shows expression around FB1 induced PCD lesions. Expression was also detected in phloem and in young trichomes. 6) Using transient expression assays in tobacco, and in stable transformed A. thaliana lines, it was shown that MC1 interacts in vivo with the negative regulator of cell death LSD1. Coimmunoprecipitation experiments in double transgenic lines overexpressing both MC1 and LSD1 could not confirm these results. 7) Transgenic A. thaliana plants overexpressing MC1 C-terminal fused to YFP were screened for YFP expression. Several independent lines showed subcellular localisation of MC1 in cytoplasm and nucleus.